The replication behavior of Saccharomyces cerevisiae DNA in human cells

We studied the replication of random genomic DNA fragments from Saccharomyces cerevisiae in a long-term assay in human cells. Plasmids carrying large yeast DNA fragments were able to replicate autonomously in human cells. Efficiency of replication of yeast DNA fragments was comparable to that of similarly sized human DNA fragments and better than that of bacterial DNA. This result suggests that yeast genomic DNA contains sequence information needed for replication in human cells. To examine whether DNA replication in human cells would initiate specifically at a yeast origin of replication, we monitored initiation on a plasmid containing the yeast 2-micron autonomously replicating sequence (ARS) in yeast and human cells. We found that while replication initiates at the 2-micron ARS in yeast, it does not preferentially initiate at the ARS in human cells. This result suggests that the sequences that direct site specific replication initiation in yeast do not function in the same way in human cells, which initiate replication at a broader range of sequences.by J.A. Huberman     

Replication initiates at multiple locations on an autonomously replicating plasmid in human cells.

We have used a two-dimensional gel electrophoresis mapping technique to determine where DNA replication initiates on a plasmid which utilizes a fragment of human DNA to replicate autonomously in human cells. Replication was found to initiate at multiple locations on the plasmid carrying the human sequence, in contrast to the pattern seen for an Epstein-Barr virus vector which served as a control with a fixed origin. The family of repeats, a portion of the Epstein-Barr virus origin of replication which is present our plasmid, was shown to function as a replication fork barrier. The nature of the stalled replicative intermediates on the human DNA-based plasmid further indicated that replication did not initiate at a single fixed position each time the plasmid replicated. The results suggest that the replication apparatus used to duplicate DNA in human cells may not have precise sequence requirements which target initiation to specific locations.     

Human claspin is a ring-shaped DNA-binding protein with high affinity to branched DNA structures

Claspin is an essential protein for the ATR-dependent activation of the DNA replication checkpoint response in Xenopus and human cells. Here we describe the purification and characterization of human Claspin. The protein has a ring-like structure and binds with high affinity to branched DNA molecules. These findings suggest that Claspin may be a component of the replication ensemble and plays a role in the replication checkpoint by directly associating with replication forks and with the various branched DNA structures likely to form at stalled replication forks because of DNA damage.     

Human cytomegalovirus UL44 concentrates at the periphery of replication compartments, the site of viral DNA synthesis

The formation of replication compartments, the subnuclear structures in which the viral DNA genome is replicated, is a hallmark of herpesvirus infections. The localization of proteins and viral DNA within human cytomegalovirus replication compartments is not well characterized. Immunofluorescence analysis demonstrated the accumulation of the viral DNA polymerase subunit UL44 at the periphery of replication compartments and the presence of different populations of UL44 in infected cells. In contrast, the viral single-stranded-DNA binding protein UL57 was distributed throughout replication compartments. Using "click chemistry" to detect 5-ethynyl-2'-deoxyuridine (EdU) incorporation into replicating viral DNA and pulse-chase protocols, we found that viral DNA synthesis occurs at the periphery of replication compartments and that replicated viral DNA subsequently localizes to the interior of replication compartments. The interiors of replication compartments also contain regions in which UL44 and EdU-labeled DNA are absent. The treatment of cells with a viral DNA polymerase inhibitor reversibly caused the dispersal of both UL44 and EdU-labeled viral DNA from replication compartments, indicating that ongoing viral DNA synthesis is necessary to maintain the organization of replication compartments. Our results reveal a previously unappreciated complexity of the organization of human cytomegalovirus replication compartments.     

Autonomous DNA replication in human cells is affected by the size and the source of the DNA.

We previously developed short-term and long-term assays for autonomous replication of DNA in human cells. This study addresses the requirements for replication in these assays. Sixty-two random human genomic fragments ranging in size from 1 to 21 kb were cloned in a prokaryotic vector and tested for their replication ability in the short-term assay. We found a positive correlation between replication strength and fragment length, indicating that large size is favored for efficient autonomous replication in human cells. All large fragments replicated efficiently, suggesting that signals which can direct the initiation of DNA replication in human cells are either very abundant or have a low degree of sequence specificity. Similar results were obtained in the long-term assay. We also used the same assays to test in human cells a random series of fragments derived from Escherichia coli chromosomal DNA. The bacterial fragments supported replication less efficiently than the human fragments in the short-term and long-term assays. This result suggests that while the sequence signals involved in replication in human cells are found frequently in human DNA, they are uncommon in bacterial DNA.     

The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G(1)/S border and throughout the S phase and was negligible in both G(0) and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase alpha. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an approximately 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.     

Simian virus 40 origin- and T-antigen-dependent DNA replication with Drosophila factors in vitro.

DNA replication of double-stranded simian virus 40 (SV40) origin-containing plasmids, which has been previously thought to be a species-specific process that occurs only with factors derived from primate cells, is catalyzed with an extract derived from embryos of the fruit fly Drosophila melanogaster. This reaction is dependent upon both large T antigen, the SV40-encoded replication initiator protein and DNA helicase, and a functional T-antigen binding site at the origin of DNA replication. The efficiency of replication with extracts derived from Drosophila embryos is approximately 10% of that observed with extracts prepared from human 293 cells. This activity is not a unique property of embryonic extracts, as cytoplasmic extracts from Drosophila tissue culture cells also support T-antigen-mediated replication of SV40 DNA. By using highly purified proteins, DNA synthesis is initiated by Drosophila polymerase alpha-primase in a T-antigen-dependent manner in the presence of Drosophila replication protein A (RP-A; also known as single-stranded DNA-binding protein), but neither human RP-A nor Escherichia coli single-stranded DNA-binding protein could substitute for Drosophila RP-A. In reciprocal experiments, however, Drosophila RP-A was able to substitute for human RP-A in reactions carried out with human polymerase alpha-primase. These results collectively indicate that many of the specific functional interactions among T antigen, polymerase alpha-primase, and RP-A are conserved from primates to Drosophila species. Moreover, the observation that SV40 DNA replication can be performed with Drosophila factors provides a useful assay for the study of bidirectional DNA replication in Drosophila species in the context of a complete replication reaction.     

Isolation of the viral DNA replication complex from adeno-associated virus type 1-infected cells.

The replication complex active in adeno-associated virus type 1 (AAV-1) DNA synthesis in vitro was solubilized, with a nonionic detergent, from the nuclei of human embryonic kidney cells coinfected with AAV-1 and an early temperative-sensitive mutant (ts125) of human adenovirus type 5 at the nonpermissive temperature (40.5 degrees C). The complex sedimented with a mean size of 23S and contained parental AAV-1 DNA. Most of the DNA synthesized with the AAV-1 DNA replication complex in vitro was AAV-1 DNA, as revealed by DNA-DNA hybridization and sedimentation in a neutral sucrose gradient. However, it sedimented in an alkaline sucrose gradient as molecules smaller than AAV-1 DNA (14.4S). The AAV-1 DNA replication complex was not formed in cells infected with AAV-1 alone.     

Distinct populations of human PCNA are required for initiation of chromosomal DNA replication and concurrent DNA repair

The integrity of genomic DNA during the cell division cycle in eukaryotic cells is maintained by regulated chromosomal DNA replication and repair of damaged DNA. We have used fractionation and reconstitution experiments to purify essential factors for the initiation of human chromosomal DNA replication in late G1 phase template nuclei from human cells. Here, we report the identification of soluble PCNA as an essential initiation factor in this system. Recombinant histidine-tagged human PCNA can substitute for purified endogenous human PCNA to initiate human chromosomal DNA replication. It is recruited specifically to discrete DNA replication foci formed during initiation in vitro. The template nuclei also contain DNA breaks as result of the synchronisation procedure. A separate population of chromatin-bound PCNA is already present in these template nuclei at discrete DNA damage foci, co-localising with gamma-H2AX, RPA and Rad51. This DNA damage-associated PCNA population is marked by mono-ubiquitination, suggesting that it is involved in DNA repair. Importantly, the population of damage focus-associated PCNA is neither involved in, nor required for, the initiation of chromosomal DNA replication in the same nuclei.     

Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system

Initiation of DNA replication is tightly controlled during the cell cycle to maintain genome integrity. In order to directly study this control we have previously established a cell-free system from human cells that initiates semi-conservative DNA replication. Template nuclei are isolated from cells synchronized in late G₁ phase by mimosine. We have now used DNA combing to investigate initiation and further progression of DNA replication forks in this human in vitro system at single molecule level. We obtained direct evidence for bidirectional initiation of divergently moving replication forks in vitro. We assessed quantitatively replication fork initiation patterns, fork movement rates and overall fork density. Individual replication forks progress at highly heterogeneous rates (304 ± 162 bp/min) and the two forks emanating from a single origin progress independently from each other. Fork progression rates also change at the single fork level, suggesting that replication fork stalling occurs. DNA combing provides a powerful approach to analyse dynamics of human DNA replication in vitro.     

Mouse DNA primase plays the principal role in determination of permissiveness for polyomavirus DNA replication.

We have investigated the species-specific replication of polyomavirus DNA in the cell-free system that was established previously (Y. Murakami, T. Eki, M. Yamada, C. Prives, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 83:6347-6351, 1986). Extracts from various species of cells supported polyomavirus DNA replication in a species-specific manner that was consistent with the host range specificity of polyomavirus; extracts prepared from mouse and hamster cells were active, whereas extracts prepared from human, monkey, and insect cells were inactive. The addition of DNA polymerase alpha-primase purified from mouse cells induced the replication of polyomavirus DNA in a cell-free system containing polyomavirus large tumor antigen and nonpermissive cell extracts, such as human and insect cell extracts. Isolated mouse DNA primase alone also induced polyomavirus DNA replication in human cell extracts but not in insect cell extracts, indicating that mouse DNA primase plays the principal role in determining permissiveness for polyomavirus DNA replication.     

Mutation of cyclin/cdk phosphorylation sites in HsCdc6 disrupts a late step in initiation of DNA replication in human cells

Cyclin-dependent kinases (Cdk) are essential for promoting the initiation of DNA replication, presumably by phosphorylating key regulatory proteins that are involved in triggering the G1/S transition. Human Cdc6 (HsCdc6), a protein required for initiation of DNA replication, is phosphorylated by Cdk in vitro and in vivo. Here we report that HsCdc6 with mutations at potential Cdk phosphorylation sites was poorly phosphorylated in vitro by Cdk, but retained all other biochemical activities of the wild-type protein tested. Microinjection of mutant HsCdc6 proteins into human cells blocked initiation of DNA replication or slowed S phase progression. The inhibitory effect of mutant HsCdc6 was lost at the G1/S transition, indicating that phosphorylation of HsCdc6 by Cdk is critical for a late step in initiation of DNA replication in human cells.     

De novo DNA methylation at nonrandom founder sites 5' from an unmethylated minimal origin of DNA replication in latent Epstein-Barr virus genomes

Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well-characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells.     

The Mammalian DNA Replication Elongation Checkpoint: Implication of Chk1 and Relationship with Origin Firing as Determined by Single DNA Molecule and Single Cell Analyses

The regulation of DNA replication initiation is well documented, for both unperturbed and damaged cells. The regulation of elongation, or fork velocity, however, has only recently been revealed with the advent of new techniques allowing us to view DNA replication at the single cell and single DNA molecule levels. Normally in S phase, the progression of replication forks and their stability are regulated by the ATR-Claspin-Chk1 pathway. We recently showed that replication fork velocity varies across the human genome in normal and cancer cells, but that the velocity of a given fork is positively correlated with the distance between origins on the same DNA fiber. Accordingly, in DNA replication-deficient Bloom’s syndrome cells, reduced fork velocity is associated with an increased density of replication origins. Replication elongation is also regulated in response to DNA damage. In human colon carcinoma cells treated with the topoisomerase I inhibitor camptothecin, DNA replication is inhibited both at the level of initiation and at the level of elongation through a Chk1-dependent checkpoint mechanism. Together, these new findings demonstrate that replication fork velocity (fork progression) is coordinated with inter-origin distance and that it can be actively slowed down by Chk1-dependent mechanisms in response to DNA damage. Thus, we propose that the intra-S phase checkpoint consist of at least three elements: (1) stabilization of damaged replication forks; (2) suppression of firing of late origins; and (3) arrests of normal ongoing forks to prevent further DNA lesions by replication of a damaged DNA template.     

PD184352 releases the regular hypoxic reversible DNA replication arrest in T24 cells

The oxygen dependent regulation of DNA replication is an essential property of proliferating mammalian cells. In human T24 bladder cancer cells, several hours of hypoxia leads to reversible DNA replication arrest and re-entry of oxygen induces a burst of replication initiation. This short communication provides strong evidence that PD184352 initiates DNA replication in living hypoxic cells without elevating the oxygen level. PD184352 releases the regular hypoxic replicon arrest, however, at a low intensity compared to the effect of reoxygenation. Moreover, PD184352 shows no effect on normoxically incubated as well as reoxygenated T24 cells.     

Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.     

The human beta-globin replication initiation region consists of two modular independent replicators

Previous studies have shown that mammalian cells contain replicator sequences, which can determine where DNA replication initiates. However, the specific sequences that confer replicator activity were not identified. Here we report a detailed analysis of replicator sequences that dictate initiation of DNA replication from the human beta-globin locus. This analysis suggests that the beta-globin replication initiation region contains two adjacent, redundant replicators. Each replicator was capable of initiating DNA replication independently at ectopic sites. Within each of these two replicators, we identified short, discrete, nonredundant sequences, which cooperatively determine replicator activity. Experiments with somatic cell hybrids further demonstrated that the requirements for initiation at ectopic sites were similar to the requirements for initiation within native human chromosomes. The replicator clustering and redundancy exemplified in the human beta-globin locus may account for the extreme difficulty in identifying replicator sequences in mammalian cells and suggest that mammalian replication initiation sites may be determined by cooperative sequence modules.     

Characteristics of DNA replication in the long-term culture of human cells at the stationary phase

Peculiarities of DNA replication in cultured human diploid fibroblasts in logarithmic and stationary phases were studied using DNA autoradiography. The rate of DNA replication fall from 30-36 mu/hour at active proliferative phase to 18-20 mu/hour at late stationary phase. This phenomenon is characteristic of stationary cultures after stimulation to proliferate by changing medium as well as by culturing without stimulation. Possible mechanisms of DNA replication rate alteration in senescent human cells are discussed.